Prevention of aflatoxin in pistachios1

Awareness that undesirable levels of aflatoxin in food and feed may have
serious consequences for human and animal health is increasing. Aflatoxins, probably the
most studied and widely known mycotoxins, were first noted in the early 1960s. They are
among the most potent mutagenic substances known; there is extensive experimental and
epidemiological evidence that they induce liver cancer (WHO, 1998). The term aflatoxin
refers to a class of chemical compounds of related structure; among these aflatoxin B1
is considered to be the most potent carcinogen.

The major aflatoxin-producing fungi are Aspergillus flavus and Aspergillus
parasiticus. Under favourable temperature and humidity conditions these fungi grow on
certain foodstuffs, most commonly groundnuts, dried fruit, tree nuts (almonds, pecans,
walnuts, pistachios and Brazil nuts), spices and a range of cereals (especially maize).
Production of aflatoxin is optimal at relatively high temperatures, so contamination is
most acute and widespread in warm, humid climates. Although contamination is generally
considered to be a problem in tropical and subtropical regions of Africa, Asia and Latin
America, aflatoxins have also been found in temperate countries of Europe and North
America (FAO, 1979, 1982).

CONTAMINATION

Fungal contamination and subsequent production of aflatoxin can occur in
crops in the field, at harvest, during postharvest operations and in storage. The rate and
degree of contamination are dependent on temperature, humidity and soil and storage
conditions. Prevention, particularly by excluding or reducing toxigenic mould growth and
toxin production in susceptible food crops, is the most effective way to restrict
mycotoxin contamination. In practice, this can be accomplished by reducing fungal
infections in growing crops through rapid drying and correct storage of the harvested
crops, the use of effective anti-mould preservatives and adherence to proper postharvest
processing, transport and distribution practices.

Many mycotoxins, including aflatoxins, can form during the growing stages
of certain crops. Climate, sources of fungal inoculum (or suitability of the fungal
substrate), potential insect vectors and the plant response (or the plant susceptibility
to fungal infestation) can interact to result in specific mycotoxin occurrence. Damage
caused by insects can expose susceptible tissues to colonization by toxigenic fungi with
subsequent mycotoxin formation.

Following harvest and during shipment and storage of agricultural
commodities, toxigenic mould growth and potential mycotoxin production are influenced by
many factors including moisture level, temperature, aeration, infestation by insects and
other microorganisms, storage time, chemical treatments, spore infection density and
storage conditions (especially leakage of water or condensation).

By far the most critical environmental factors determining whether a
substrate will support mould growth are moisture content, temperature and time. Thus,
drying, proper storage and suitable transportation are of prime importance in prevention.

The prevention of mycotoxin contamination in storage is largely a matter
of strict moisture control of the crop. There must be no insect activity, as insects can
create favourable microclimates for toxigenic fungal growth; no moisture migration; no
condensation or water leaks; and no rodent activity, as the moisture level could be
increased by urination. In summary, conditions which restrict fungal growth will almost
invariably limit or exclude mycotoxin production.

Where harvesting occurs in dry weather, mycotoxin contamination does not
usually reach alarming proportions. It becomes a problem where harvesting is done in very
humid weather. In many developing countries, the combination of insufficient drying
equipment coupled with humid atmospheric conditions results in unacceptable levels of
aflatoxin in harvested groundnuts, tree nuts and other foods.

AFLATOXIN IN PISTACHIOS

What conditions favour mould growth and aflatoxin production in
pistachio nuts and how can this be prevented? Many Aspergillus species infect nuts
and cause decay of the kernels before harvest. For example, it was reported that up to 13
species were isolated from pistachio kernels from orchards in Iran and 14 species from
orchards in the United States (California) (Doster and Michailides, 1994a).

The shells of most pistachio nuts split naturally in the orchard prior to
harvest. Fortunately, the hull covering the shell usually remains intact, protecting the
kernel from invasion by moulds and insects (see Figure). Nuts that are poorly protected by
hulls are most prone to contamination in the orchard. Sometimes the hull is attached to
the shell so that it splits with the shell, exposing the kernel to moulds and insects.
This is called an "early split". In some countries the proportion of early
splits can be as high as 30 percent. In a study done in the United States, approximately 1
to 5 percent of the nuts were early splits. When early splits were examined in one study
about 20 percent of the samples (50 nuts each) were found to be contaminated with
aflatoxin, while there was no contamination in nuts with hulls that remained intact in the
orchard (Doster and Michailides, 1995; Sommer, Buchanan and Fortlage, 1986). Although the
importance of early splitting for mould, aflatoxin and insect contamination is well
established, very little is known concerning when early splits occur and become
contaminated. This knowledge could aid in timing the application of either chemical or
biological control treatments.

Besides early splitting, hull rupture may occur from very late
harvesting, bird damage and cracking. The navel orange worm (Amyelois transitella)
commonly infests nuts with ruptured hulls and such infestation has been associated with
very high levels of aflatoxin. Nuts infested with navel orange worm pose less of a danger
because they are easier to eliminate by sorting, and consumers are likely to reject them.

Early split nuts that are not infected in the orchard may become infected
during transport and handling. High humidity and high temperature within bulk bins provide
ideal conditions for the infection of early split nuts, which dramatically increases the
incidence and level of aflatoxin contamination, until nuts are mycologically stabilized by
drying or refrigeration.

The development of Aspergillus mould in pistachio litter has been
related to the increase of Aspergillus inoculum in the orchard and results in
greater numbers of mouldy and mycotoxin-contaminated pistachio nuts. Certain practices
such as burying or removing litter may decrease colonization by Aspergillus moulds
in the orchard. Another possible treatment for pistachio litter includes the application
of microorganisms that limit the development of Aspergillus mould. Further research
is needed to determine if any of these practices would reduce Aspergillus
colonization of litter enough to decrease the numbers of mouldy and mycotoxin-contaminated
nuts harvested (Doster and Michailides, 1994b).

Pistachio nut and shell within the hull

RECOMMENDATIONS OF THE CODEX ALIMENTARIUS COMMISSION

The Codex Recommended International Code of Hygienic Practice for Tree
Nuts (CAC/RCP 6-1972) (FAO/WHO, 1994b) provides basic hygienic requirements for orchard,
farm processing (shelling and hulling) and commercial shelling and in-shell operations. It
covers environmental sanitation in growing and food production areas, sanitary harvesting,
transportation, plant facilities and operating requirements, operating practices and
production requirements, in particular raw material handling, inspection and sorting,
preparation, processing and storage. Prevention and control measures should allow
compliance with the Codex standard for unshelled pistachio nuts (CODEX STAN 131-1981)
(FAO/WHO, 1994a) as well as with respective provisions for food additives and contaminants
laid down in Codex texts.

GUIDELINES FOR PREVENTING MOULD CONTAMINATION
AND AFLATOXIN PRODUCTION

Harvesting

- Tree nuts, including pistachios, should be harvested as soon as possible
after maturation to avoid quality loss and to minimize problems involving fungal attack
and infestation with insects, especially the navel orange worm.
- Signs of optimum harvesting time are: separation or ease of separation of the hull from
the shell; decrease in fruit removal force; drying of hull and kernel.
- Pistachios should not be shaken to the ground to avoid contamination of open shells by
litter and soil.
- In hand picking of young trees, clean tarps should be used to avoid contact of the nuts
with soil and litter.

Postharvest handling

- Pistachios should be hulled and dried soon after harvest to minimize
shell staining and decay and to ensure safety. If temporary storage of fresh pistachios at
the dehydration plant is necessary, they should be cooled and held before hulling at 0°C
and relative humidity lower than 70 percent.
- Pistachios should be sorted before cold storage to remove defective nuts, leaves, twigs
and other foreign materials.
- Fumigation with methyl bromide or phosphine can be used to control insects in stored
nuts. It may be necessary to repeat fumigation periodically depending on the conditions
and duration of storage.

Drying

- In the two-stage process, the hulled nuts are first dried for about
three hours in a column dryer or a rotating drum dryer (air temperature around 82°C) to
reach kernel moisture of 12 to 13 percent. Nuts are then transferred to a flat-bottomed
grain bin (second stage) where they continue drying with warm air (temperature less than
49°C) for one to two days to reach a moisture content of 5 to 6 percent.
- In the single-stage process, a self-unloading bin dryer is used at air temperature of
60° to 66°C. Drying time is eight hours.

Postdrying storage

- Dried nuts are usually stored in bins, silos or other bulk storage
containers for a few weeks or several months before final processing and preparation for
market.
- Optimum storage conditions of 10°C or lower and 65 to 70 percent relative humidity must
be maintained to minimize deterioration, including the growth of Aspergillus flavus.
- Protection against insects is also essential during the storage period.

Processing

- Nuts are sorted, using visual or light reflectance electronic sorting
techniques, to eliminate those with defects (nuts with adhering hulls, stained and mouldy
nuts).
- Empty or partially empty pistachios are separated by an air stream, and unsplit
pistachios by floatation in water.
- Shells are cracked for extraction of kernels when desired.
- Nuts are sorted to eliminate those with possible aflatoxin contamination.
- Nuts are salted, flavoured and roasted.
- Nuts are packaged: in-shell nuts, shelled nuts and nut meat (broken kernels) in various
types and sizes.

Postprocessing storage

- Water activity should be kept below 0.70 at 25°C (moisture content less
than 7 percent).
- Relative humidity should be kept below 70 percent to avoid any significant mould growth.
- Temperature should be kept between 0° and 10°C depending on expected storage duration.
The lower the temperature, the longer the storage life.
- Low oxygen (less than 0.5 percent) helps maintain flavour quality and effects insect
control. Exclusion of oxygen is usually done by vacuum packaging or by packaging in
nitrogen.

MAXIMUM LEVELS

The need for setting maximum levels of aflatoxins in foods and feeds is
generally recognized. Several countries, particularly some industrialized ones, have
already set specific regulations. Limits for aflatoxin B1 in foodstuffs range
from 0 to 30 µg/kg, while those for total aflatoxins range from 0 to 50 µg/kg (FAO,
1997). In 1994-1995, FAO, in collaboration with the Dutch National Institute of Public
Health and Environment, carried out a global survey of mycotoxin regulations which covered
some 90 countries (FAO, 1997). The survey indicated that only France, Greece and Jordan
have set specific regulations for aflatoxin levels in pistachio nuts (Table 1).

Table 1
Maximum tolerable level of aflatoxin in pistachio

Country

Aflatoxin

Level (µg/kg)

France

B1

1

Greece

B1

5

B1 +B2
+G1 +G2

10

Jordan

B1

15

B1 +B2
+G1 +G2

30

Source: FAO, 1997.

In several countries, the maximum tolerable level for aflatoxin in
pistachios is covered under the category "nut products" (Table 2). In 36 other
countries, pistachio nuts are covered under the category "all foods".

Table 2
Maximum tolerable level of aflatoxin in nut products

Country

Aflatoxin

Level (µg/kg)

Australia

B1 +B2
+G1 +G2

15

Canada

B1 +B2
+G1 +G2

15

Cyprus

B1

5

(to be confirmed)

B1 +B2
+G1 +G2

10

Israel

B1

5

B1 +B2
+G1 +G2

15

New Zealand

B1 +B2
+G1 +G2

15

Philippines

B1 +B2
+G1 +G2

20

United Kingdom

B1 +B2
+G1 +G2

4

Source: FAO, 1997.

The setting of internationally agreed maximum tolerable levels for
aflatoxins in food and feed is of global importance. Unfortunately, establishing such
levels is difficult because of the divergent views on the necessary level of protection
between producing and consuming countries. Despite years of work carried out on this
subject by various Codex committees, very little has been achieved.

EVALUATIONS OF THE JOINT FAO/WHO EXPERT COMMITTEE ON FOOD ADDITIVES

The Joint FAO/World Health Organization (WHO) Expert Committee on Food
Additives (JECFA) is the international scientific advisory body responsible for the safety
evaluation of naturally occurring toxic substances such as aflatoxins. JECFA provides the
independent scientific advice that forms the basis for the development of food safety
standards by the Codex Alimentarius Commission. JECFA reviewed aflatoxin toxicity in 1987
and concluded that there was insufficient information to establish a tolerable level of
exposure. It recommended that the level of aflatoxin in food should be reduced to "as
low as reasonably achievable" (ALARA) (WHO, 1998). ALARA is defined as the
concentration of a substance that cannot be eliminated from a food without involving the
loss of the food altogether or without severely compromising the availability of major
food supplies.

In 1996 and 1997, JECFA re-evaluated aflatoxins. At its June 1997
meeting, the committee concluded from a quantitative risk assessment on aflatoxins that
there is no significant difference in risk to human health between the maximum levels of
10 µg/kg and 20 µg/kg for aflatoxin B1 in food (WHO, 1998). The Codex
Committee on Food Additives and Contaminants has responsibility for proposing guideline
levels for naturally occurring toxicants in foods and feeds. It will examine the JECFA
recommendation, and its proposals will then be reviewed by the Codex Alimentarius
Commission for possible adoption as international standards.

From a regulatory standpoint, aflatoxins are considered unavoidable
contaminants in foods since they cannot be prevented or eliminated by current good
agricultural practices. National regulations should not jeopardize the availability offood
crops at reasonable prices, and the implications for farming, especially subsistence
farming in developing countries, must also be considered. Thus exposure of the population
to some level of aflatoxins is tolerated.

The total value of pistachio nuts traded in the international market
exceeds US$500 million (Moaven, 1998). Harmonization of regulations at the global level
helps importing countries to secure regular supplies ofcommodities and helps
exporting countries to find markets for their products. A balance must be sought between
the needs of importing and exporting countries, without jeopardizing the consumer's
health. The setting of unnecessarily low guideline levels for aflatoxin may result in a
technical barrier to trade.

FAO MYCOTOXIN PREVENTION PROGRAMMES

According to FAO estimates, up to 25 percent of the world's food
production is lost at postharvest level. Such losses include those caused by mould and
mycotoxin contamination. FAO is greatly concerned with the impact of mycotoxin
contamination of foods because of its negative effects on human health, food trade and
food availability and consumption. For years, FAO's Food Quality and Standards Service has
provided the Organization's member countries with advice on policies and strategies for
preventing and controlling mycotoxin contamination; its activities include efforts to
harmonize mycotoxin tolerance levels in foods and feeds and respective methods of sampling
and analysis.

The implementation of prevention programmes may include not only the
prevention of mycotoxin formation in agricultural products (through better farm management
practices, use of fungus- and toxin-resistant varieties, proper irrigation and fertilizer
practices, pest control, crop rotation, improved postharvest techniques - including drying
and storage practices - and adequate use of chemicals), but also their removal through
detoxification or decontamination. Also required are routine surveillance; regulatory
measures to control the flow of mycotoxin-contaminated commodities in national and
international trade; and information, education and communication activities. Each of
these approaches forms a part of an overall strategy to minimize the impact of mycotoxin
contamination.

In dealing with mycotoxin problems, FAO has organized, in collaboration
with WHO and the United Nations Environment Programme (UNEP), international meetings where
experts exchange their views, identify priorities at the regional or international level
and propose strategies for further action. Two joint FAO/WHO/UNEP international
conferences on mycotoxins have been held, in 1977 and 1987. Some recommendations from the
last conference referred specifically to prevention measures, such as:

- the development of feasible and practical methods to prevent preharvest
contamination of food crops and to improve postharvest practices at both local and
large-warehouse levels, so as to prevent fungal infection and insect damage;
- the coordination of research activities on issues pertaining to mycotoxin toxicity,
incidence, prevention and elimination;
- the development of feasible, economical and safe means of decontamination of
mycotoxin-contaminated food commodities and products.

FAO has also organized regional meetings in order to review and adapt the
proposed international strategy. Workshops were held in Asia (1990), in Africa (1991) and
in Latin America (1991) to assist in identifying specific regional needs and priorities.
The Asian workshop, for instance, referred to the need for a bold Asian initiative for the
1990s to observe the "decade of prevention and control" with activities designed
to minimize mycotoxin contamination in food.

At the national level, FAO has assisted a number of developing countries in
strengthening their capabilities to prevent and control the contamination of foods by
mycotoxins. These activities have been carried out through field projects addressing
specific problems related to mycotoxin prevention, surveillance and control. Of the 200
projects in FAO's Special Action Programme for the Prevention of Food Losses, some 50 have
included components for mycotoxin control. For example, the Southeast Asia project,
involving 13 countries, introduced appropriate postharvest technologies related to
mycotoxin prevention, with emphasis on drying facilities.

FAO promotes the dissemination of technical information on
mycotoxin-related issues. Several publications and training aids on various aspects of
mycotoxin prevention programmes, including methods of sampling and analysis, have been
prepared and distributed widely. A training syllabus was developed in 1990 for use in
short-term courses on aflatoxin analysis in Africa. Publications created as part of
project activities include a directory of mycotoxin prevention and control institutions in
Asia. A global compilation of maximum tolerable levels of mycotoxins in foods and feeds
has been recently issued (FAO, 1997).

CURRENT NEEDS

Aflatoxin contamination of pistachio nuts is undoubtedly a serious
problem for many producing countries. The European Union's 1997 ban on pistachio imports
from Iran because of high levels of aflatoxin in some shipments exemplifies the
seriousness of this problem. Unless concrete measures are taken by the pistachio-producing
countries and by traders to address the problem, the situation may worsen. Prevention of
mould growth and of aflatoxin production throughout the pistachio production and
distribution chain is the key solution. The systematic and organized application of
integrated phytosanitary management (IPSM) in orchards to eliminate, if not reduce, mould
infection during the cultivation stage, coupled with a Hazard Analysis and Critical
Control Point (HACCP) approach to prevent contamination during the harvest and postharvest
stages, should lead to the desired results (Kader, 1992). This will not be possible
without proper training of all actors in the chain: farmers, processors, warehouse
keepers, traders and transporters. Food control officials and extension workers should be
closely involved in this effort.

Further research is needed to explore other means of preventing mould
infection and aflatoxin production. These could include prevention of contamination in the
field through the use of biological control agents that are pathogenic to Aspergillus
flavus, such as yeast or harmless bacteria, and that could act as ecologically
friendly bio-competitors (FAO, 1979; Katz, 1996). Scientists are exploring a genetically
engineered antibody to toxin production which could be applied when conditions are
favourable to toxin production. In other words, the mould would grow but would not produce
toxin. Finally, research is needed to produce pistachio varieties that are more resistant
to aflatoxin.

On the regulatory side, efforts should be made to establish, as soon as
possible, internationally agreed guideline levels for aflatoxin in pistachio nuts which
would be accepted by all parties so as to eliminate possible technical barriers to trade.
Such guideline levels should be based on sound scientific evidence and on realistic risk
assessment. The recent JECFA work on this subject is a breakthrough and provides hope for
a positive development in the near future.

Summary/Résumé/Resumen

Prevention of aflatoxin in
pistachios

Aflatoxin contamination in food and
feed supplies may have serious consequences for human and animal health. Aflatoxins are
commonly associated with groundnuts, dried fruit, tree nuts, spices and cereals.
Contamination is most acute and widespread in the warm, humid areas of Africa, Asia and
Latin America. However, aflatoxins are found in temperate areas of North America and
Europe as well. Contamination can occur in crops in the field, at harvest, during
postharvest operations and in storage. The rate and degree of aflatoxin contamination are
dependent on temperature, humidity and soil and storage conditions.
Early splitting, hull rupture and damage from birds and insects make pistachios
susceptible to contamination. Research is needed to prevent mould infection and aflatoxin
production and to produce pistachio varieties that are more resistant to aflatoxin.
Prevention of mould growth and of aflatoxin production is crucial and requires proper
training of farmers, processors, warehouse keepers, traders and transporters. Food control
officials and extension workers should be closely involved in these efforts. The Codex
Alimentarius Recommended International Code of Hygienic Practice for Tree Nuts provides
basic hygienic requirements for orchard, farm processing (shelling and hulling) and
commercial shelling and in-shell operations. Prevention and control measures should allow
compliance with the Codex standard for unshelled pistachio nuts (Codex Standard 131-1981).
From a regulatory standpoint, aflatoxins are considered unavoidable contaminants in foods
since they cannot be prevented or eliminated by current good agricultural practices. Thus
exposure of the population to some level of mycotoxins is tolerated. The need for setting
maximum levels of aflatoxins in foods and feeds is generally recognized. Several
countries, particularly some industrialized ones, have already set specific limits,
ranging from 0 to 30 µg/kg for aflatoxin B1 in foodstuffs and from 0 to 50
µg/kg for total aflatoxins.
The setting of internationally agreed maximum tolerable levels for aflatoxins in food and
feed is of global importance. Recently, the Joint FAO/WHO Expert Committee on Food
Additives (JECFA) concluded that there is no significant difference in risk to human
health between the maximum levels of 10 µg/kg and 20 µg/kg for aflatoxin B1 in
food. Unnecessarily low guideline levels for aflatoxin may result in a technical barrier
to trade.